Claims:1. An extracorporeal device (100) for absorption of endotoxins and cytokines from a biological fluid, wherein the extracorporeal device comprises of:
an outer shell (102) with an inlet (108) to allow inflow of an impure biological fluid and an outlet (110) to allow outflow of a purified biological fluid;
an active inner core (104) comprising of a plurality of narrow spun rod-shaped fibres (112) with a filler incorporated polymer matrix is arranged vertically inside the outer shell (102), wherein the active inner core (104) facilitates absorption of endotoxins and cytokines; and
a resin assembly (106) to allow the polymer fibre matrix at both ends of the extracorporeal device (100) to be stably fixed inside the outer shell (102) by using a biocompatible resins.
2. The extracorporeal device (100) as claimed in claim 1, wherein the biological fluid comprises of blood or plasma.
3. The extracorporeal device (100) as claimed in claim 1, wherein the plurality of narrow spun rod-shaped fibres (112) are capable of adsorbing at least one of endotoxins, cytokines or both.
4. The extracorporeal device (100) as claimed in claim 1, wherein the cytokines comprises of HMGB1 and IL-1 ß.
5. The extracorporeal device (100) as claimed in claim 1, wherein the polymer matrix is a biocompatible polymer selected from a group comprising at least one of thermoplastic polyurethane (TPU), polylactic acid or polycaprolactone.
6. The extracorporeal device (100) as claimed in claim 1, wherein the filler material comprises of:
an endotoxin absorbent, wherein the endotoxin absorbent comprises at least one of cationic material such as polymyxin B sulphate or chitosan;
a cytokine absorbent, wherein the cytokine absorbent comprises at least one of anionic material such as poly (sodium 4-strenesulfonate) or zirconium hydrogen phosphate;
7. The extracorporeal device (100) as claimed in claim 1, wherein the biocompatible resins of the resin assembly (106) comprises at least one of epoxy resins or polyurethane, wherein the thickness of biocompatible resins is in the range of 1-2mm.
8. The extracorporeal device (100) as claimed in claim 1, wherein the outer shell (102) is made of material possessing chemical resistance, thermal stability, rigidity and transparency, wherein the material comprises at least one of biocompatible polycarbonate or polyester-based material.
9. The extracorporeal device (100) as claimed in claim 1, wherein the outer shell (102) maximizes the active surface of the biological fluid in an aseptic condition and reduces turbulence in the flow.
10. A method for synthesis of fibres (112) that adsorb endotoxins and cytokines, wherein the method comprises of:
a) dissolving Thermoplastic Polyurethane in a mixture of dimethyl formamide and dimethyl sulfoxide in a ratio of 1:1 or 11:9 by stirring at a temperature in the range of 50-55ºC for 5-8 hours to obtain a Thermoplastic Polyurethane solution;
b) reducing the temperature of Thermoplastic Polyurethane solution to a temperature in the range of 35-40 ºC followed by mixing 5-10% of Poly (sodium 4-styrene sulfonate) by constant stirring for 5-15 minutes;
c) reducing the temperature of the resultant reaction mixture to a temperature in the range of 25-30ºC followed mixing with 0.1-5 wt% of Polymyxin b Sulphate by constant stirring for 5-10 minutes to obtain an opaque solution;
d) passing the obtained homogenous polymer solution via a narrow spinneret through a coagulant bath comprising a endotoxin-free water for a time period of 30-60 seconds at 22-28ºC to obtain a solid narrow fibres; and
e) spinning the obtained solid narrow fibres and drying at a temperature in the range of 30-35ºC for 72 to 96 hrs.
11. The method as claimed in claim 10, wherein the concentration of Thermoplastic Polyurethane solution is 0.25 to 0.30 g/ml.
12. A method for synthesis of fibres (112) that adsorb endotoxins, wherein the method comprises of:
a) dissolving Thermoplastic Polyurethane in a mixture of dimethyl formamide and dimethyl sulfoxide in a ratio of 1:1 or 11:9 by stirring at a temperature in the range of 50-55oC for a time period of 5-8 hours;
b) transforming the Thermoplastic Polyurethane dissolved matrix at a temperature in the range of 25-30oC, adding 0.1-5% of Polymyxin b Sulphate and resultant solution is stirred for a time period of 10-15mins;
c) passing the dissolved polymer matrix filler solution through a narrow spinneret to obtain a polymer fibre;
d) passing the obtained polymer fibre through a cooling region comprising of an endotoxin free water solution for a time period of 30-60 seconds to obtain the solid narrow fibres; and
e) spinning the obtained solid narrow fibres and dried at a temperature in the range of 30-35 ºC for a time period of 72 -96 hrs.
13. The method as claimed in claim 12, wherein the concentration of Thermoplastic Polyurethane is in the range of 0.25-.30 g/ml and the concentration of Polymyxin b Sulphate is in the range of 0.1-5 wt% to Thermoplastic Polyurethane.
14. A method for synthesis of fibres (112) that absorb cytokines, wherein the method comprises of:
a) dissolving Thermoplastic Polyurethane in a mixture of dimethyl formamide and dimethyl sulfoxide in a ratio of 1:1 or 11:9 by stirring at a temperature in the range of 50-55oC for a time period of 5-8 hours;
b) transforming the Thermoplastic Polyurethane dissolved matrix at a temperature in the range of 35-40oC, adding 5-10% of powered Poly (sodium 4-styrenesulfonate) and resultant solution is stirred for a time period of 5-15mins;
c) passing the dissolved polymer matrix filler solution through a narrow spinneret to obtain a polymer fibre;
d) passing the obtained polymer fibre through a cooling region comprising of an endotoxin free water solution for a time period of 30-60 seconds to obtain a solid narrow fibres; and
e) spinning the obtained solid narrow fibres and drying at a temperature in the range 30-35 ºC for a time period of 72 -96 hrs.
15. The method as claimed in claim 14, wherein the concentration of Thermoplastic Polyurethane is in the range of 0.25-.30 g/ml.
16. The method as claimed in claims 10, 12 and 14, wherein the narrow spinneret is a 5 ml glass syringe.

Dated this 29th day of June 2021

Signature

Harish Naidu
Patent Agent (IN/PA-2896)
Agent for the Applicant
, Description:FIELD OF INVENTION
[0001] Embodiments of the present invention relates to a field of extracorporeal devices. More particularly, it relates to an extracorporeal device and method for absorption of endotoxins and cytokines (HMGB1 and IL-1 ß) from plasma and blood to treat bacterial sepsis.

BACKGROUND OF THE INVENTION

[0002] Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Intensive care units (ICU) of hospitals are facing a serious problem with the appearance of increased numbers of sepsis patients. Estimated mortality from sepsis of Gram-negative aetiology ranges from 30 to 50 percent of the overall total number of septic deaths.

[0003] The world is seeing an unprecedented threat from bacterial infections at present. Gram-negative bacteria produce its deleterious effect through the production of potent endotoxins that give rise to Cytokine storm and potentially life-threatening complications like septic shock and multi-organ failure. The lack of an early diagnostic test or effective treatment process enhances the risk associated with the sepsis.

[0004] Conventional therapy such as antibiotics and surgical procedures is crucial for treating sepsis, but these approaches cannot succeed to reverse the effects of the bacterial toxins and Cytokines already released into the blood. In the past decade, several devices/therapies were developed to remove these toxins from sepsis patients. Unfortunately, none of them was successful. Toraymyxin 20-R and a Polymyxin B-immobilized cartridge are one of such devices that allows endotoxin removal to control sepsis progression However, this was not considered to be a complete success because only particular subgroups of patients greatly benefitted from its usage as evidenced through numerous random controlled trials.

[0005] Similarly, Cytosorb is another device that removes both pro and anti-inflammatory Cytokines by adsorption in sepsis patients. However, Cytosorb therapy did not change the systemic cytokine plasma levels and also it has not kept the anti-inflammatory cytokines IL-4 and 10 to down-regulate the TLR signalling, which leads to pro-inflammatory cytokine synthesis.

[0006] Hence, other than the currently available endotoxin/cytokine removal devices which suffer from various practical shortcomings, there is a need for an improved, more specific, cost effective and advanced device, which can control sepsis is highly demanding to address the aforementioned issues.

SUMMARY OF THE INVENTION
[0007] In accordance with an embodiment of the invention, an extracorporeal device for absorption of endotoxins and cytokines from a biological fluid is disclosed. The extracorporeal device comprises of an outer shell with an inlet to allow inflow of an impure biological fluid and an outlet to allow outflow of a purified biological fluid, an active inner core comprising of a plurality of narrow spun rod-shaped fibres with a filler incorporated polymer matrix is arranged vertically inside the outer shell, wherein the active inner core facilitates absorption of endotoxins and cytokines and a resin assembly to allow the polymer fibre matrix at both ends of the extracorporeal device to be stably fixed inside the outer shell by using a biocompatible resins.

[0008] In accordance with an embodiment of the invention, the biological fluid comprises of blood or plasma.

[0009] In accordance with an embodiment of the invention, the plurality of narrow spun rod-shaped fibres are capable of adsorbing at least one of endotoxins, cytokines or both.

[0010] In accordance with an embodiment of the invention, the cytokines comprises of HMGB1 and IL-1 ß.

[0011] In accordance with an embodiment of the invention, the polymer matrix is a biocompatible polymer selected from a group comprising at least one of thermoplastic polyurethane (TPU), polylactic acid or polycaprolactone.

[0012] In accordance with an embodiment of the invention, the filler material comprises of an endotoxin absorbent, wherein the endotoxin absorbent comprises at least one of cationic material such as polymyxin B sulphate or chitosan and a cytokine absorbent, wherein the cytokine absorbent comprises at least one of anionic material such as poly (sodium 4-strenesulfonate) or zirconium hydrogen phosphate.

[0013] In accordance with an embodiment of the invention, the biocompatible resins of the resin assembly comprises at least one of epoxy resins or polyurethane, wherein the thickness of biocompatible resins is in the range of 1-2mm.

[0014] In accordance with an embodiment of the invention, the outer shell is made of material possessing chemical resistance, thermal stability, rigidity and transparency, wherein the material comprises at least one of biocompatible polycarbonate or polyester-based material.

[0015] In accordance with an embodiment of the invention, the outer shell maximizes the active surface of the biological fluid in an aseptic condition and reduces turbulence in the flow.

[0016] In accordance with another embodiment of the invention, a method for synthesis of fibres that adsorb endotoxins and cytokines is disclosed, wherein the method comprises of dissolving Thermoplastic Polyurethane in a mixture of dimethyl formamide and dimethyl sulfoxide in a ratio of 1:1 or 11:9 by stirring at a temperature in the range of 50-55ºC for 5-8 hours to obtain a Thermoplastic Polyurethane solution, reducing the temperature of Thermoplastic Polyurethane solution to a temperature in the range of 35-40 ºC followed by mixing 5-10% of Poly (sodium 4-styrene sulfonate) by constant stirring for 5-15 minutes, reducing the temperature of the resultant reaction mixture to a temperature in the range of 25-30ºC followed mixing with 0.1-5 wt% of Polymyxin b Sulphate by constant stirring for 5-10 minutes to obtain an opaque solution, passing the obtained homogenous polymer solution via a narrow spinneret through a coagulant bath comprising a endotoxin-free water for a time period of 30-60 seconds at 22-28ºC to obtain a solid narrow fibres and spinning the obtained solid narrow fibres and drying at a temperature in the range of 30-35ºC for 72 to 96 hrs.

[0017] In accordance with an embodiment of the invention, the concentration of Thermoplastic Polyurethane solution is 0.25 to 0.30 g/ml.

[0018] In accordance with another embodiment of the invention, a method for synthesis of fibres that adsorb endotoxins, wherein the method comprises of dissolving Thermoplastic Polyurethane in a mixture of dimethyl formamide and dimethyl sulfoxide in a ratio of 1:1 or 11:9 by stirring at a temperature in the range of 50-55oC for a time period of 5-8 hours, transforming the Thermoplastic Polyurethane dissolved matrix at a temperature in the range of 25-30oC, adding 0.1-5% of Polymyxin b Sulphate and resultant solution is stirred for a time period of 10-15mins, passing the dissolved polymer matrix filler solution through a narrow spinneret to obtain a polymer fibre, passing the obtained polymer fibre through a cooling region comprising of an endotoxin free water solution for a time period of 30-60 seconds to obtain the solid narrow fibres and spinning the obtained solid narrow fibres and dried at a temperature in the range of 30-35 ºC for a time period of 72 -96 hrs.

[0019] In accordance with an embodiment of the invention, wherein the concentration of Thermoplastic Polyurethane is in the range of 0.25-.30 g/ml and the concentration of Polymyxin b Sulphate is in the range of 0.1-5 wt% to Thermoplastic Polyurethane.

[0020] In accordance with another embodiment of the invention, a method for synthesis of fibres that absorb cytokines, wherein the method comprises of dissolving Thermoplastic Polyurethane in a mixture of dimethyl formamide and dimethyl sulfoxide in a ratio of 1:1 or 11:9 by stirring at a temperature in the range of 50-55oC for a time period of 5-8 hours, transforming the Thermoplastic Polyurethane dissolved matrix at a temperature in the range of 35-40oC, adding 5-10% of powered Poly (sodium 4-styrenesulfonate) and resultant solution is stirred for a time period of 5-15mins, passing the dissolved polymer matrix filler solution through a narrow spinneret to obtain a polymer fibre, passing the obtained polymer fibre through a cooling region comprising of an endotoxin free water solution for a time period of 30-60 seconds to obtain a solid narrow fibres and spinning the obtained solid narrow fibres and drying at a temperature in the range 30-35 ºC for a time period of 72-96 hrs.

[0021] In accordance with an embodiment of the invention, the concentration of Thermoplastic Polyurethane is in the range of 0.25-.30g/ml.

[0022] In accordance with an embodiment of the invention, the narrow spinneret is a 5ml glass syringe.

[0023] To further clarify the advantages and features of the present disclosure, a more particular description of the disclosure will follow by reference to specific embodiments thereof, which are illustrated in the appended figures. It is to be appreciated that these figures depict only typical embodiments of the disclosure and are therefore not to be considered limiting in scope. The disclosure will be described and explained with additional specificity and detail with the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The disclosure will be described and explained with additional specificity and detail with the accompanying figures in which:

[0025] FIG. 1a is a extracorporeal device for absorption of endotoxins and cytokines from biological fluids, in accordance with an embodiment of the present invention.

[0026] FIG. 1b is a cross-sectional view of an active core of the extracorporeal device for absorption of endotoxins and cytokines from biological fluids, in accordance with an embodiment of the present invention.

[0027] FIG. 2 is a flow-chart illustrating method steps for synthesis of fibres that adsorb endotoxins and cytokines, in accordance with an embodiment of the present invention.

[0028] FIG. 3 is a flow-chart illustrating method steps for synthesis of fibres that adsorb endotoxins, in accordance with an embodiment of the present invention.

[0029] FIG. 4 is a flow-chart illustrating method steps for synthesis of fibres that absorb cytokines, in accordance with an embodiment of the present invention.

[0030] FIG. 5 is a graph illustrating endotoxin adsorption in human plasma, in accordance with an embodiment of the present invention.

[0031] FIG. 6 is a graph illustrating HMGB1 adsorption in human plasma, in accordance with an embodiment of the present invention.

[0032] FIG. 7 is a graph illustrating IL-1 ß adsorption in human plasma, in accordance with an embodiment of the present invention.

[0033] Further, those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and may not have necessarily been drawn to scale. Furthermore, in terms of the method steps, chemical compounds, and parameters used herein may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the figures with details that will be readily apparent to those skilled in the art having the benefit of the description herein.

DETAILED DESCRIPTION OF THE INVENTION
[0034] For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Such alterations and further modifications in the illustrated system, and such further applications of the principles of the disclosure as would normally occur to those skilled in the art are to be construed as being within the scope of the present disclosure.

[0035] The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.

[0037] In the following specification and the claims, reference will be made to a number of terms, which shall be defined to have the following meanings. The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0038] As used herein “Endotoxin” are complex lipopolysaccharides (LPS) which form an inherent fraction of the outer cell wall of all gram-negative bacteria. Endotoxin is structurally a Lipopolysaccharide (LPS) which is an important structural component of the Gram-negative bacteria. LPS is released into the environment during multiplication as well as the death of the bacteria, act as endotoxins and is a very potent immune-modulatory molecule, producing a strong immune response in vertebrates. Endotoxin causes abnormal activation of the host immune system, leading to host cell injury followed by multiple organ failure, which is the ultimate cause of the death of sepsis patients. Though role of endotoxin in mediating all these events is not completely clear but it has been shown that a higher concentration of endotoxin in the blood of sepsis patients correlates with a higher mortality rate and it is shown that the removal of endotoxin from blood circulation will improve the outcome of Gram-negative sepsis.

[0039] As used herein “HMGB1” a damage associated molecular patterns, functionally a Cytokine and considered as a late mediator of sepsis.

[0040] As used herein “IL-1ß” is a pro-inflammatory cytokine actively involved in the induction of TLR signalling that leads to cytokine storm in septic shock. The pro-inflammatory properties of IL-1 ß are enhanced by its binding with HMGB1 and form circulating HMGB1/ IL-1ß complex.

[0041] FIG. 1a is a extracorporeal device (100) for absorption of endotoxins and cytokines from biological fluids, in accordance with an embodiment of the present invention. FIG. 1b is a cross-sectional view of an active core (104) of the extracorporeal device (100) for absorption of endotoxins and cytokines from biological fluids, in accordance with an embodiment of the present invention. In accordance with an embodiment of the invention, an extracorporeal device (100) for absorption of endotoxins and cytokines from a biological fluid is disclosed.

[0042] According to an embodiment of the present invention, the extracorporeal device comprises of an outer shell (102) with an inlet (108) to allow inflow of an impure biological fluid and an outlet (110) to allow outflow of a purified biological fluid, an active inner core (104) comprising of a plurality of narrow spun rod-shaped fibres (112) with a filler incorporated polymer matrix is arranged vertically inside the outer shell (102). More particularly, the inner core is composed of narrow spun rod-shaped solid fibre/filament/advanced hollow fibres, which contain a filler incorporated polymer matrix.

[0043] According to an embodiment of the present invention, the active inner core (104) facilitates absorption of endotoxins and cytokines and a resin assembly (106) to allow the polymer fibre matrix at both ends of the extracorporeal device (100) to be stably fixed inside the outer shell (102) by using a biocompatible resins. Further the active inner core (104) comprises of void space (114).

[0044] According to an embodiment of the present invention, the biological fluid comprises of blood or plasma.

[0045] According to an embodiment of the present invention, the plurality of narrow spun rod-shaped fibres (112) are capable of adsorbing at least one of endotoxins, cytokines or both.

[0046] According to an embodiment of the present invention, the cytokines comprises of HMGB1 and IL-1 ß.

[0047] According to an embodiment of the present invention, the polymer matrix is a biocompatible polymer selected from a group comprising at least one of thermoplastic polyurethane (TPU), polylactic acid or polycaprolactone.

[0048] According to an embodiment of the present invention, the filler material comprises of an endotoxin absorbent, wherein the endotoxin absorbent comprises at least one of cationic material such as polymyxin B sulphate or chitosan and a cytokine absorbent, wherein the cytokine absorbent comprises at least one of anionic material such as poly (sodium 4-strenesulfonate) or zirconium hydrogen phosphate, wherein it will effectively remove HMGB1 which in turn binds to IL-1 ß using its cytokine binding domain and thereby effectively removes the cytokines (HMGB1 and IL-1 ß).

[0049] According to an embodiment of the present invention, the extracorporeal device (100) of the present invention makes use of the affinity of the DNA binding domains of HMGB1 towards the anionic surface. Further, the polymer composite of the present invention is in the form of narrow fibres (112) which can be produced through phase inversion methods such as wet or dry-wet or electrospinning. The purpose of the outer shell (102) is to maximize the active surface of the blood in an aseptic condition and to reduce the turbulence in the flow.

[0050] According to an embodiment of the present invention, the biocompatible resins of the resin assembly (106) comprises at least one of epoxy resins or polyurethane, wherein the thickness of biocompatible resins is in the range of 1-2mm. Overall, the resin assembly (106) acts as a proof sealing in the extracorporeal device (100) to offer excellent barrier property, dimensional stability, biocompatibility and proper adhesion.

[0051] According to an embodiment of the present invention, the outer shell (102) is made of material possessing chemical resistance, thermal stability, rigidity and transparency, wherein the material comprises at least one of biocompatible polycarbonate or polyester-based material.

[0052] According to an embodiment of the present invention, the outer shell (102) maximizes the active surface of the biological fluid in an aseptic condition and reduces turbulence in the flow. Further the outer shell (102) can be manufactured by 3D printed or other molded processes.

[0053] According to an embodiment of the present invention, the extracorporeal device (100) of the present invention provides a combined approach in eliminating both endotoxin and cytokine (HMGB1 and IL-1 ß) molecules from plasma or blood and thereby controls sepsis.

[0054] FIG. 2 is a flow-chart illustrating method steps for synthesis of fibres that adsorb endotoxins and cytokines, in accordance with an embodiment of the present invention. The method comprises of at step 202, dissolving Thermoplastic Polyurethane in a mixture of dimethyl formamide and dimethyl sulfoxide in a ratio of 1:1 or 11:9 by stirring at a temperature in the range of 50-55ºC for 5-8 hours to obtain a Thermoplastic Polyurethane solution, wherein the concentration of Thermoplastic Polyurethane solution is 0.25 to 0.30 g/ml.

[0055] Next at step 204, reducing the temperature of Thermoplastic Polyurethane solution to a temperature in the range of 35-40 ºC followed by mixing 5-10% of Poly (sodium 4-styrene sulfonate) by constant stirring for 5-15 minutes. At step 206, reducing the temperature of the resultant reaction mixture to a temperature in the range of 25-30ºC followed mixing with 0.1-5 wt% of Polymyxin b Sulphate by constant stirring for 5-10 minutes to obtain an opaque solution.

[0056] Next at step 208, passing the obtained homogenous polymer solution via a narrow spinneret through a coagulant bath comprising a endotoxin-free water for a time period of 30-60 seconds at 22-28ºC to obtain a solid narrow fibres, wherein the narrow spinneret is a 5 ml glass syringe. And finally at step 210, spinning the obtained solid narrow fibres and drying at a temperature in the range of 30-35ºC for 72 to 96 hrs.

[0057] FIG. 3 is a flow-chart illustrating method steps for synthesis of fibres that adsorb endotoxins, in accordance with an embodiment of the present invention. The method steps comprises of at step 302, dissolving Thermoplastic Polyurethane in a mixture of dimethyl formamide and dimethyl sulfoxide in a ratio of 1:1 or 11:9 by stirring at a temperature in the range of 50-55oC for a time period of 5-8 hours, wherein the concentration of Thermoplastic Polyurethane is in the range of 0.25-.30 g/ml and the concentration of Polymyxin b Sulphate is in the range of 0.1-5 wt% to Thermoplastic Polyurethane.

[0058] At step 304, transforming the Thermoplastic Polyurethane dissolved matrix at a temperature in the range of 25-30oC, adding 0.1-5% of Polymyxin b Sulphate and resultant solution is stirred for a time period of 10-15mins. Next at step 306, passing the dissolved polymer matrix filler solution through a narrow spinneret to obtain a polymer fibre, wherein the narrow spinneret is a 5 ml glass syringe.

[0059] At step 308, passing the obtained polymer fibre through a cooling region comprising of an endotoxin free water solution for a time period of 30-60 seconds. And at step 310, spinning the obtained solid narrow fibres and dried at a temperature in the range of 30-35ºC for a time period of 72 -96 hrs.

[0060] FIG. 4 is a flow-chart illustrating method steps for synthesis of fibres that absorb cytokines, in accordance with an embodiment of the present invention. The method step comprises of at step 402, dissolving Thermoplastic Polyurethane in a mixture of dimethyl formamide and dimethyl sulfoxide in a ratio of 1:1 or 11:9 by stirring at a temperature in the range of 50-55oC for a time period of 5-8 hours, wherein the concentration of Thermoplastic Polyurethane is in the range of 0.25-.30 g/ml.

[0061] At step 404, transforming the Thermoplastic Polyurethane dissolved matrix at a temperature in the range of 35-40oC, adding 5-10% of powered Poly (sodium 4-styrenesulfonate) and resultant solution is stirred for a time period of 5-15mins. And at step 406, passing the dissolved polymer matrix filler solution through a narrow spinneret to obtain a polymer fibre, wherein the narrow spinneret is a 5 ml glass syringe.

[0062] Next at step 408, passing the obtained polymer fibre through a cooling region comprising of an endotoxin free water solution for a time period of 30-60 seconds to obtain a solid narrow fibres and finally at step 410, spinning the obtained solid narrow fibres and drying at a temperature in the range 30-35 ºC for a time period of 72-96 hrs.

EXPERIMENTAL RESULTS

[0063] The endotoxin, HMGB1 and IL-1 ß adsorption efficiency of fibres were determined by calculating the difference between initial and final (after incubation with fibre) concentrations of these additives in Plasma/blood. The sorption characteristics of endotoxin and Cytokines (HMGB1 and IL-1 ß) on the polymer composite narrow fibres is analysed at different conditions such as endotoxin and Cytokine concentration, adsorbent concentration, surface area, time interval, etc. It is observed that fibres absorb a known concentration of endotoxin and Cytokines (HMGB1 and IL-1 ß) from plasma completely within 3 hours of incubation.
Invitro Circulation Experiment
[0064] According to an embodiment of the present invention, invitro circulation experiments were carried out using plasma or blood spiked with a known concentration of Endotoxin Units (EU)/mL (40 EU/mL), HMGB1 (50,000 nanograms/mL) and IL-1 ß (50,000 picograms/mL) with a known surface area of fibre at 25-30ºC at a flow rate of 50-200 mL/minutes.

[0065] The samples were collected at 1hour intervals until 3 hours. The remaining endotoxin level of plasma and blood measured by Limulus Amoebocyte Lysate Kinetic QCL Kit. The remaining HMGB1 levels in the supernatant has been determined Indirect ELISA using Anti-HMGB1 antibodies. The remaining IL-1 ß has been estimated by Sandwich ELISA by using human IL-1 ß ELISA Kit.

[0066] The fibres adsorbed Endotoxin, HMGB1 and IL-1 ß from human plasma and blood. It is observed that the fibres with a known surface area adsorbed 10,000 Endotoxin Units (EU) from plasma/blood within 3 hours at room temperature. Fig 5 represents a graph illustrating endotoxin adsorption in human plasma, in accordance with an embodiment of the present invention.

[0067] FIG. 6 is a graph illustrating HMGB1 adsorption in human plasma, in accordance with an embodiment of the present invention. It is observed that fibres with a known surface area completely adsorbed HMGB1 of concentration 50,000 nanograms/mL from plasma/blood. From Fig 6, it is evident that the maximum adsorption occurred from 0-2 hours of incubation.

[0068] FIG. 7 is a graph illustrating IL-1 ß adsorption in human plasma, in accordance with an embodiment of the present invention. The fibres with a known surface area adsorbed 50,000 picograms/mL of IL-1 ß from plasma/blood. From Fig 7, it is evident that the adsorption happened maximum from 2 to 3 hours followed by 1-2 hours and 0-1 hours. Hence it is assumed that IL-1 ß adsorption mainly happened after HMGB1 adsorption is complete or started. It may be also noted that the HMGB1 bound fibres will act as the filler for IL-1 ß binding and removal.

EXAMPLES OF SYNTHESIS OF NARROW SPUN ROD-SHAPED FIBRES (112) WITH A FILLER INCORPORATED POLYMER MATRIX

Synthesis of fibres to adsorb Endotoxins, HMGB1 and IL-1 ß

[0069] The synthesis comprises steps of dissolving Thermoplastic Polyurethane (TPU) in the mixture of dimethyl formamide and Dimethyl sulfoxide in ratio of 1:1 or 11:9 by sufficient stirring at 50-55 ºC for 5-8 hours to obtain the Thermoplastic Polyurethane solution, wherein the concentration of Thermoplastic Polyurethane solution is 0.25 to 0.30 g/ml.

[0070] Next, the temperature of Thermoplastic Polyurethane solution is reduced to 35-40 ºC followed by mixing 5-10% of Poly (sodium 4-styrene sulfonate) by constant stirring for 5-15 minutes. Followed by the same, the temperature of the resultant reaction mixture was reduced to 25-30ºC and mixed with 0.1-5 wt% of Polymyxin b Sulphate by constant stirring for 5-10 minutes to get an opaque solution, wherein this solution is used for the synthesis of fibres that adsorbs endotoxin, HMGB1 and IL-1 ß from plasma and blood.

[0071] After the mixing, the well distributed and well dispersed homogenous polymer solution is passed through a narrow spinneret, which is a 5 ml glass syringe or similar through a coagulant bath such as endotoxin-free water for 30-60 seconds at 22-28ºC. In this stage, the bath removes the solvent and make the polymer in solid fibres form. These solid narrow fibres were spun and were dry at 30-35ºC for 72 to 96 hrs or until they get dried.

Synthesis of fibres to adsorb Endotoxin, and fibres that adsorb HMGB1 and IL-1 ß separately

Synthesis of fibres that adsorb Endotoxin
[0072] The Thermoplastic Polyurethane (TPU) was dissolved in the mixture of Dimethyl formamide and Dimethyl sulfoxide in ratio of 1:1 or 11:9 by stirring at 50-55oC for 5-8 hours. The Thermoplastic Polyurethane dissolved matrix is transformed at 25-30oC, then added 0.1-5% percentage Polymyxin b Sulphate and solution was further stirred for 10-15mins.

[0073] The concentration of Thermoplastic Polyurethane was fixed at 0.25-.30 g/ml and Polymyxin b Sulphate contents was 0.1-5 wt% to Thermoplastic Polyurethane. The dissolved polymer matrix filler solution is pushed through a spinneret, which is a 5 ml glass syringe. The polymer fibre passed through a cooling region such endotoxin free water solution around 30-60 seconds. After the spinning the fibre were dry at 30-35 ºC for 72 -96 hrs.

Synthesis of fibres that adsorb HMGB1 and IL-1 ß
[0074] The Thermoplastic Polyurethane (TPU) was dissolved in the mixture of Dimethyl formamide and Dimethyl sulfoxide in ratio of 1:1 or 11:9 by stirring at 50-55oC for 5-8 hours. The Thermoplastic Polyurethane dissolved matrix is transformed at 35-40oC, then added 5-10% powered Poly (sodium 4-styrenesulfonate) and solution was further stirred for 5-15min.

[0075] The concentration of Thermoplastic Polyurethane was fixed at 0.25-.30 g/ml. The dissolved polymer matrix filler solution was pushed through a spinneret, which is a 5 ml glass syringe. The polymer fibre passed through a cooling region such endotoxin free water solution around 30-60 seconds. After the spinning the fibre were dry at 30-35 ºC for 72 -96 hrs.

[0076] Various embodiments of the present invention provide an extracorporeal device (100) with a horizontal inlet valve (108) and outlet valve (110) for biological fluids such as plasma and blood for detoxifying endotoxins, HMGB1 and IL1- ß. The extracorporeal device (100) of the present invention achieves simultaneous removing of endotoxins, HMGB1 and IL1- ß by selective adsorption from plasma and blood to treat bacterial sepsis.

[0077] The extracorporeal device (100) of the present invention employs polymer matrix and fillers which are biocompatible polymer composite fibrous, wherein the polymer matrix comprises of thermoplastic polyurethane, Polylactic acid or Polycaprolactone and fillers can be Polymyxin B sulphate or Chitosan and Poly (sodium 4-styrenesulfonate), Zirconium hydrogen phosphate or other cation exchange materials.

[0078] The present invention uses fibres developed by dry wet spinning method of 0.1-1mm diameter was used as the active surface of device for Endotoxins, HMGB1 and IL1- ß from plasma and blood.

[0079] Also, the extracorporeal device (100) of the present invention uses 0.1-5 % Polymyxin b sulphate as the adsorbent for endotoxin removal plasma and blood, uses a negatively charged fiber surface for the selective binding and removal of HMGB1 plasma and blood, uses 5-10 % Poly (sodium 4-styrenesulfonate) as the HMGB1 adsorbent to remove HMGB1 from plasma and blood, uses HMGB1 bound polymer fibre surface as the adsorbent for IL1- ß removal.

[0080] Overall, the extracorporeal device (100) of the present invention provides simultaneous elimination of endotoxins and cytokines (HMGB1 and IL-1 ß) to achieve an effective approach in sepsis control. Since the endotoxin removal stops the endotoxin mediated cytokine storm in sepsis condition and further removal of HMGB1 stops the TLR signalling that leads to pro-inflammatory cytokine synthesis, wherein the HMGB1 is a basic amino acid rich protein and it is removed from the system by the use of an anionic surface-based polymer.

[0081] Further, the removal of HMGB1 in turn helps in the removal of IL-1 ß achieved by the cytokine-binding domain of HMGB1. The polymer matrix based fibres synthesized via the wet spinning method acts as an adsorbent to bind and remove endotoxins, HMGB1 and IL-1 ß. Therefore, the extracorporeal device (100) of the present invention effectively removes LPS, HMGB1 and IL-1 ß molecules from the blood and regulates the associated inflammatory responses in sepsis.

[0082] While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended. As would be apparent to a person skilled in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein.

[0083] The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts need to be necessarily performed. Also, those acts that are not dependant on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.